Alan S. Waggoner - US grants

Our objective in the proposed research is to develop optical probes (fluorescent dyes and chromophores) that can be used to (1) measure membrane potentials in nerve tissue and cells that are too small for microelectrode studies, (2) measure intracellular pH, (3) measure cellular free calcium, and (4) identify transformed cells and quantitate the antigenic makeup of cell surfaces. We also plan to determine the molecular mechanism by which these new probes carry out their functions. We intend to accomplish our objectives by careful design, synthesis characterization and evaluation of the new optical probes. Evaluation will be carried out with biological and model systems. Much effort will be directed toward development of the highly fluorescent and absorptive polymethine dyes as optical probes of biological systems.

A video image memory system, acquistion procesor and digitizer, and a cursor generator will be acquired to complete a distributed, yet integrated, imaging facilty for the Center for Fluorescence Research in Biomedical Sciences. Interactive video image acquisition and analysis, and supermini computers for data acquisition and reduction are also part of the imaging facility. Four separate laboratories are networked to allow multiple user applications. A wide variety of projects in cell biology, cell physiology, developmental biology, regulatory biology, and biological chemistry will be supported by in the instrumentation. Among the projects to be supported are analyses of the growth factor stimulation of quiescent cells and macrophage chemotaxis. Ratio imaging will be used as a spectroscopic method for determining the spatial and temporal changes in (cellular) physiological parameters such as pH and pCa. Multiple parameter analysis of two or more separable fluorescent probes will also be used to correlate spatial and temporal dynamics of a variety of cellular funcitons. Three dimensional reconstruction will be carried out on actin networks in living cells. A method of two wavelength total internal reflection image analysis will be used to quantitate the distance from the cell substrate to selected molecules. Methods for mapping the spatial and temporal variations in fluorescence lifetime and fluorescence anisotropy of suitably labeled proteins in living cells will be developed. The first target for mapping with be calmodulin. Overall, the equipment, coupled with other resources in the Center, will permit state-of-the-art measurements greatly extending our understanding of cellular function.

9318891 Taylor The vision of the Center for Light Microscope Imaging andBiotechnology (CLMIB) is that understanding the cellular and molecular mechanisms of fundamental biological processes is within reach during the next decade. This will be made possible by integration of three major tools: biochemistry, molecular biology/genetics, and optical techniques which allow exploration of the in vivo dynamics of cell structure and chemistry. The third of these is the focus of the Center. The Center's strengths are in development of, a) novel reagents for detecting, measuring and manipulating the chemical and molecular components of cells, b) new and integrated modalities of light microscopy to detect and measure temporal and spatial changes in cell structure and chemistry, and c) advanced computing methods to record, analyze, display and model complex imaging data. Technique and instrument development is driven by the needs of biological research to answer basic questions in cell and developmental biology, and by emerging applications in biotechnology. The Center's research and training activities are organized into programs in Cell Biology, Developmental Biology, Applied Biotechnology, Fluorescence-Based Reagents, Machine-Vision Microscopy, 3-D Microscopy, Advanced Image Analysis, and Automated Interactive Microscopy. Twenty-five faculty from 6 departments and 26 graduate students from 7 departments are now working on research projects that involve collaborations between biologists, chemists, physicists, engineers and computer scientists. Direct or indirect support from the Center has resulted in 162 publications. A new interdisciplinary course, Applications of Fluorescence Spectroscopy in Biological Research, was initiated, and a biotechnology training laboratory is being constructed for this and related courses. Other knowledge transfer activities include organizing two conferences, supporting a general-use imaging laboratory for outside researchers, and cr eating a scientist exchange program with industrial collaborators. Transfer of technology occurs through interactions with 14 corporations that license the Center's technology and/or collaborate on research projects. These corporations include multi-nationals such as Dupont, Kodak, Carl Zeiss and Procter and Gamble, as well as biotechnology start-ups such as Biological Detection Systems, One-Cell Systems and Cadus Pharmaceuticals. Two new corporations have already been formed in Western Pennsylvania based on technology transfer from the STC. Two patents have issued from Center research, 4 patents have been submitted, and 27 disclosures have been submitted to the University. The STC is involved in two major K-12 outreach projects: a) a group of high school students and teachers worked with the Center for 6 weeks in the summer in a program called "Careers in Applied Science and Technology (CAST), and b) the Center is collaborating with the Carnegie Science Center in Pittsburgh and the Studio of Creative Inquiry at Carnegie Mellon University to produce a planetarium show called "Journey to the Center of the Cell". This project will use advanced visualization technologies, based on work in the Center, to educate the general public, particularly young students, about scientific discovery and the biology of the living cell.***

This proposal is for the purchase of a fluorescence- activated cell sorter to replace one purchased in 1983 at the inception of the Center for Fluorescence Research in Biomedical Sciences at Carnegie Mellon University. The PI and co-PI are leaders in the flow cytometry community and the current FACS has been used in 38 research publications in cell biology in the 9 years since its installation. No other instrument exists at CMU. Limitations in its capabilities compared to newer instruments and difficulties with repairing and maintaining it justify replacement. The group of major and minor users receives over $3,290,000 in direct cost research support per year from federal and other agencies, including over $690,000 per year from NSF. The proposal replacement instrument will be used for research in a number of areas of cell, molecular and developmental biology, including membrane traffic, somatic cell genetics, fluorescent probe development, mitochondria physiology and cell differentiation and will be an integral part of the recently-established NSF Science and Technology Research Center in Light Microscopy Imaging and Biotechnology at Carnegie Mellon University

DESCRIPTION (provided by applicant): It is well known that fluorescence detection provides the basis of flow cytometry, fluorescence imaging, DNA sequencing, microarray detection, drug testing, diagnostics, and so on. It is our goal to extend the power of fluorescence detection in cancer research and diagnostics, by developing new fluorescent probes that absorb and fluoresce in the red and near infrared regions of the spectrum. These advances will: (1) increase the number of parameters that can be acquired in a single experiment (hence, more information becomes available through correlation of cell/tissue properties), (2) increase sensitivity (since cellular autofluorescence is much smaller in the red and near IR), and (3) allow imaging deeper into tissues (since absorbance and fluorescence will be away from hemoglobin absorption and in a range where light scattering and absorption is reduced). The new fluorescent reagents will be photostable, chemically stable, water soluble, brightly fluorescent and non-photo-toxic to cells and tissues. This will be accomplished by synthetic modifications in the molecular structure of cyanine (polymethine) dyes and by stabilizing dye structure through encapsulation methods including cyanine-cyclodextrin rotaxanes, nucleic acid encapsulation, sandwich complexation, and covalent self-encapsulation.

DESCRIPTION (provided by applicant): We will develop new technologies to employ quantum dots in biological imaging in vitro. In an evolving program, quantum dot technology will be developed specifically for (I) cell identification and tracking of cells in tissues, (2) tracking cell proliferation through multiple generations in tissues (3) vasculature location, and (4) deep 3-D imaging of Qdot-labeled polymer scaffold structures relative to the Qdot-labeled cells in engineered tissue models. Quantum Dots Corporation will prepare new types of Qdots that have properties suited to deep imaging of cells and structures in tissues. Particular emphasis will be placed on near infrared Qdots for imaging deeper in tissues. The Science and Technology Center at Carnegie Mellon University will then develop methods to derivatize Qdots for existing and new applications in cell biology. The STC will initially use available Qdots and conjugates, label cells by a variety of means, then test the cells for fluorescent brightness, stability of labeling, and cell survival and function. As newer quantum dots become available, they will be similarly tested and compared with existing Qdots and existing organic fluorescent probes, e.g., Alexa dyes and cyanine dyes. Fluorescence lifetime imaging capability will be added to the 3-D grating imager in the STC for enhancing signal-to-background in deep tissue imaging by taking advantage of the relatively long emission lifetime of Qdots. To obtain feedback for the development program we have included collaboration with the recently formed Bone Tissue Engineering Center at Carnegie Mellon University. In this collaboration we will examine the utility of the developing Qdot technology for studying cell location, movement and proliferation in the 3-D structures of engineered bone tissue. This is a particularly challenging and relevant system that requires Qdot technology to extend cell tracking to denser and more highly scattering tissue matrices, including hydroxyapatite-containing artificial bone matrices. By the conclusion of this project, we expect to have instrumentation and probes to perform time-resolved multicolor imaging at millimeter depth in many natural and artificial tissues. Thus the technology will be generic and have utility in many biological and medical applications.

EXPERIMENTAL AND INTEGRATED ACTIVITIES

SENSORS: Integrated Molecular Sensors Derived from Combinatorial protein and DNA Libraries

Bruce A Armitage
(EIA - 0330135)


Abstract

This research is on the construction of integrated molecular sensors derived from combinatorial libraries of proteins and DNA. An integrated molecular sensor consists minimally of two components: the recognition element that binds to the target molecule (analyte), and the signal transducer that converts the binding event into a readable signal. The recognition element will be derived from the aforementioned combinatorial libraries. These libraries will be generated either through genetic engineering for proteins or through chemical synthesis for DNA. The assumption is that at least some of the library members will fold into a three-dimensional structure that is complementary to some part of the target. Controlled in vitro selection procedures will then be used to isolate members of the libraries that bind to a particular target, namely the ricin A-chain protein, with high affinity.

The broader impacts of this research are several-fold. While much of the work proposed is of a fundamental nature, the target molecule to be used in the selections, the ricin A-chain protein is of immediate relevance. This target is important due to recently heightened concerns about the use of the ricin toxin, which includes the A-chain, in bioterrorism and biowarfare. Moreover, the methods to be developed for constructing integrated molecular sensors can be applied to many other targets. Functional sensors for use in the clinic and the field can be realized from this research. Students working on this project will be exposed to a broad range of techniques including chemical synthesis, molecular biology and optical spectroscopy. In addition to the expected academic year efforts, graduate student mentors and undergraduate students will be recruited for a special summer research team that will spend ten weeks working in a highly collaborative, multidisciplinary environment.

DESCRIPTION (provided by applicant): We propose to form a nationally visible and responsive center focused on fluorescent probe and imaging technologies for investigating pathways and networks in real time and at high resolution in living cells. The proposed Center will be formed by combining the experience and infrastructure of two already existing Centers in Pittsburgh. The research component of the Center will create a powerful toolbox of intracellular fluorescent labels and biosensors that can be used to study many, if not all, the proteins in pathways and networks of living cells. The strong fluorescent probe development program blends genetics, protein structure, nucleic acid structure and fluorescent dye chemistry. The probes we propose to generate will be genetically expressible so that exogenous macromolecules will not have to be transported into the living cells to be studied. Four superb "Driving Biology Projects" are to be part of the overall Center program in addition to the Technology Development Core. These projects are located at the University of Pittsburgh, Carnegie Mellon, Stanford and Berkeley. Their responsibility is to assist the new center in identifying the optimal technologies for development and for providing crucial feedback regarding the utility of the technologies. Imaging and informatics technologies are integrated with the new fluorescent probe technologies so that these combined tools will help cell biologists obtain large amounts of spatial and temporal information about pathways in living cells. Included in the proposal are programs for making the fluorescent probe and imaging technologies robust so that they can be provided to a wide range of cell biologists in academic research, pharmaceutical drug discovery, and biotechnology and to disseminate these technologies and provide training.

We propose to form a nationally visible and responsive center focused on fluorescent probe and imaging technologies for investigating pathways and networks in real time and at high resolution in living cells. The proposed Center will be formed by combining the experience and infrastructure of two already existing Centers in Pittsburgh. The research component of the Center will create a powerful toolbox of intracellular fluorescent labels and biosensors that can be used to study many, if not all, the proteins in pathways and networks of living cells. The strong fluorescent probe development program blends genetics, protein structure, nucleic acid structure and fluorescent dye chemistry. The probes we propose to generate will be genetically expressible so that exogenous macromolecules will not have to be transported into the living cells to be studied. Four superb "Driving Biology Projects" are to be part of the overall Center program in addition to the Technology Development Core. These projects are located at the University of Pittsburgh, Carnegie Mellon, Stanford and Berkeley. Their responsibility is to assist the new center in identifying the optimal technologies for development and for providing crucial feedback regarding the utility of the technologies. Imaging and informatics technologies are integrated with the new fluorescent probe technologies so that these combined tools will help cell biologists obtain large amounts of spatial and temporal information about pathways in living cells. Included in the proposal are programs for making the fluorescent probe and imaging technologies robust so that they can be provided to a wide range of cell biologists in academic research, pharmaceutical drug discovery, and biotechnology and to disseminate these technologies and provide training. PERFORMANCE

NIH Roadmap Initiative tag; bioimaging /biomedical imaging; fluorescent dye /probe; molecular /cellular imaging; technology /technique development

[unreadable] DESCRIPTION (provided by applicant): This proposal represents a request from a group of PHS funded investigators having overlapping imaging needs for funds to purchase a Carl Zeiss LSM 510 Meta DuoScan laser scanning confocal microscope. The system will be housed and operated by the Molecular Biosensor and Imaging Center (MBIC) as a multi-user facility in the Mellon College of Science at Carnegie Mellon University. MBIC is currently funded as one of only five National Technology Centers for Networks and Pathways with the core mission to develop optical biosensor and imaging informatics technologies for the detection of molecular-level interactions within living cells and biological tissues. The acquisition of the proposed instrument would provide on-site access to state of the art confocal microscopy instrumentation within the Mellon Institute, which is essential for a variety of live cell experiments as well as for routine analysis and characterization of the fluorescent probe technology developed within MBIC. Current instrumentation available within MBIC and partner faculty's research laboratories are both aged and unsupported or are currently fully utilized. While access to other advanced microscopy instrumentation is possible within the general Pittsburgh area, we have found that this poses a logistical problem for experiments in live cells and animals and is not practical for analyses of new biosensor and probe technologies on a routine basis. In addition, the capabilities of the proposed system meet a number of current needs that existing instrumentation is incapable of fulfilling. Since MBIC is a multi-disciplinary collaborative center which brings together a diverse collection of Carnegie Mellon faculty spanning several academic departments, colleges and research institutes, the acquired instrument will provide investigators across the university with substantially improved imaging capabilities to integrate into their research programs and will subsequently provide new opportunities to address fundamental biological questions using advanced microscopy and imaging methodologies. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): We propose to continue a nationally visible and responsive center focused on the development of novel fluorescent biosensor and detection technologies for investigating pathways and networks in real time and high spatial resolution in living cells. The renewed center retains the combined experience and infrastructure of two existing centers in Pittsburgh: the Molecular Biosensors and Imaging Center at Carnegie Mellon University and the Center for Biologic Imaging at the University of Pittsburgh. The Technology Development (Core 1) component of the center will create a powerful toolbox of intracellular fluorescent biosensors and reporters that can be used to study many, if not all, of the pathway proteins and activities in living cells. This fluorescent biosensor development program integrates efforts across multiple disciplines, including dye chemistry, molecular biology, biochemistry, structural biology, modeling, cell biology, image acquisition and analysis, and high throughput screening. We have made substantial progress in this effort during the previous 3 years of funding. Four exciting Driving Biology Projects (Core 2) are essential to the technology development effort in Core 1. These DBPs are focused on important and currently un-addressable biological problems, and will serve both as test-beds for the technology and compelling demonstrations of the value of the biosensors and reporters developed in this program. The Infrastructure (Core 3) is provided through the Center for Biologic Imaging at the University of Pittsburgh. The role of the CBI is to act as the application and outreach ami of the project as a whole by testing the new biosensors with challenging biological problems. During the last cycle of this proposal the clear mission of the CBI evolved to become the catalyst in probe implementation, and to strengthen and broaden the impact of the new probes developed by MBIC. This is acheived by providing facilities and expertise to test and validate the probes in the context of the driving biological projects, and ultimately, the biomedical research community at large. The program contains a significant technology transfer component to disseminate concepts, knowledge, software, materials, and resources to users in both academic research labs and industry. This is achieved in our Infrastructure, Training, and Dissemination cores (Core 3,4, 5) and through the technology transfer activities in the Management core (Core 6). RELEVANCE (See instructions): Understanding the regulation of proteins in networks and pathways is central to diagnosing, treating and curing a variety of human diseases. This program develops a new set of tools for observing this regulation process in live cells, as it occurs, and will lead to fundamental advances in technology, biological knowledge, and drug discovery.

DESCRIPTION (provided by applicant): The regulation of the endothelial barrier in blood vessels is a coordinated signaling process that controls the exchange of oxygen and nutrients with the surrounding tissues. Dysregulation of barrier integrity or function is implicated in a range of pathologies, including metabolic disorders such as diabetes (affecting 8.3% of the US population) and cardiovascular disorders such as atherosclerosis (affecting 25% of the US population). Regulation of the barrier is controlled by small gaseous reactive signaling molecules (e.g. nitric oxide and superoxide) that have proven challenging to detect and quantify with adequate selectivity and sensitivity in cells and more importantly in complex tissues and living animals. Imaging has provided significant insight into regulation of the barrier and related physiologic changes that correlate with disease and disease treatment. Yet imaging of signaling molecules associated with the barrier continues to pose significant challenges because the currently available fluorescent biosensors are not sufficiently specific or sensitive to directly report the concentrations and locations of the analytes. Both dye based fluorescent probes and fluorescent protein sensors suffer from limitations that prevent simultaneous correlative measurements of molecular signaling and vascular physiology. In this proposal, we develop a new class of fluorescent molecular biosensor dyes that combine the advantages of indicator dyes with the specificity of genetic encoding. By using tissue specific expression and genetically encoded subcellular targeting, these new biosensors will allow detection of Ca(II), reactive oxygen species (ROS), and reactive nitrogen species (RNS) in specific cells, at specific subcellular locations. These novel targeted fluorescent biosensors are constructed by linking together a sensitive optical sensor of Ca, ROS, or RNS with a fluorescent signaling moiety (FRET acceptor) that is activated upon binding to a genetically encoded receptor, called a fluorogen activating protein (FAP). FAP-bound sensor is able to report (fluorescence signal) the physiology of the sensing at the site of interest. Any biosensors that are not bound to the FAP target are incapable of producing a fluorescence signal and there is no background or non-specific fluorescence to complicate images or analysis of the data. The targeted biosensor dyes will be optimized to work in both cultured endothelial cells and living zebrafish. Transgenic zebrafish will be generated that express the FAP at subcellular locations in specific cells using tissue specific Cre-recombinase expression. We will use these sensors in zebrafish to assess the correlation between Ca(II), ROS and NO signaling, blood flow and barrier function. This project is a close collaboration of three Principal Investigators with distinct expertise at Carnege Mellon University and University of Pittsburgh. Dr. Bruchez is an expert on the development of multichromophore structures for biological detection, and designed the hybrid indicators for biosensing using the fluorogen activating proteins; Dr. St. Croix is an expert on endothelial cell biology RNS/ROS signaling and regulation of endothelial function and more specifically the imaging of endothelium in vitro and in vivo. Dr. Waggoner is an expert in the design of environmentally sensitive dyes, and original developer of the fluorogen activating protein technology.